Membrane transport systems are involved in a number of fundamental cellular processes yet most are poorly characterized at the structural level. The glucose transporter is a prototypic facilitative transport protein which carries out critical functions in virtually all mammalian cells. The process mediated by this family of membrane glycoproteins is the exchange of glucose between blood and cytoplasm of cells, supplying glucose for energy metabolism and the biosynthesis of sugar containing micromolecules. In addition, glucose transport in certain tissues plays a critical role in glucose homeostasis and is associated with several disease states. The long-term goal of this project is to delineate the structure of the facilitative glucose transporter and the molecular mechanism of sugar transport. The project contains two main aims. In the first aim, three residues which appear to be critical for Glut-1 transport have been identified in site-directed mutagenesis studies. Thus, the function of amino acid residues Q161 within helix 5, Q282 within helix 7, and W412 within helix 11 will be explored following expression in Xenopus oocytes of mutant transporters containing a variety of amino acid substitutions at these sites. These detailed studies form a paradigm for the characterization of additional, critical amino acids to be identified in the second aim. In addition, HXT-2, a yeast homologue of the mammalian glucose transporter, will be used to identify suppressor sites that complement mutations at these critical positions in an attempt to identify interacting sites within the glucose transporter. In the second aim, cysteine-scanning mutagenesis will be used in conjunction with the substituted cysteine accessibility method and chemical crosslinking to examine the structure of 9 of the putative transmembrane segments that have not been subjected to this analysis to date. These experiments will help identify amino acid residues that are directly involved in the mechanism of transport and will provide information about the relative orientation and bundling of transmembrane alpha helices.